Preparation method and application of colchicine-loaded polydopamine nanoparticles

Abstract

The invention relates to a preparation method and application of colchicine-loaded polydopamine nano-particles, belongs to the technical field of nano-drug delivery, and solves one of the problems of serious sudden drug release, poor targeting property, low bioavailability, large toxic and side effects and the like of an existing colchicine nano-carrier preparation. Comprising the following steps: preparing a macrophage membrane suspension and a platelet membrane suspension for later use; mixing the macrophage membrane suspension, the platelet membrane suspension and the hollow polydopamine nanoparticles to obtain a first mixed solution, and performing ultrasonic treatment on the first mixed solution to obtain a membrane-coated first mixed solution; dispersing the first mixed solution subjected to film coating treatment into a colchicine aqueous solution to obtain a second mixed solution, performing ultrasonic treatment on the second mixed solution, and loading colchicine into hollow polydopamine nanoparticles by utilizing an electrostatic adsorption effect; and at normal temperature, carrying out centrifugal treatment on the second mixed solution subjected to ultrasonic treatment, discarding the supernate, and collecting the precipitate, namely the colchicine-loaded polydopamine nano-particles.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery technology, and in particular to a method for preparing and applying polydopamine nanoparticles loaded with colchicine. Background Technology

[0002] Atherosclerosis (AS) is the primary pathological basis of most cardiovascular diseases, characterized by chronic inflammation and lipid deposition in the blood vessel walls. Colchicine is the only anti-inflammatory drug approved by the US FDA, and studies have shown that it can play a positive role in the treatment of atherosclerosis by inhibiting the activation of inflammasomes. However, colchicine itself has poor water solubility, a short half-life in vivo, lacks specific targeting, and has serious dose-dependent toxic side effects (such as bone marrow suppression and gastrointestinal reactions), which severely limit its widespread clinical application.

[0003] Nanotechnology provides a new strategy for improving the efficacy and safety of colchicine. Currently, the nanocarriers used to load colchicine mainly include liposomes and liposome-like carriers (such as phospholipid-cholesterol liposomes and alcohol carriers), polymer nanoparticles (such as PLGA nanoparticles and chitosan nanoparticles), and inorganic nanocarriers (such as mesoporous silica nanoparticles and phosphorylated MSNs). However, these carriers have the following common problems: (1) limited drug loading efficiency and easy drug burst release, making it difficult to achieve continuous treatment; (2) poor biocompatibility of the carrier itself, or the degradation products may be toxic; (3) lack of active targeting ability to the microenvironment of atherosclerotic plaques (such as high expression of macrophages and platelet aggregation), resulting in insufficient drug concentration at the lesion site and high exposure to non-target tissues, which aggravates toxic side effects.

[0004] Polydopamine (PDA) is a biocompatible and biodegradable natural derivative polymer. Its hollow structure (HPDA) possesses a high specific surface area and pore volume, making it ideal for loading drug molecules. Furthermore, the surface of PDA is rich in catechol and amino groups, facilitating functionalization, and its structure is unstable in acidic environments, making it an ideal carrier for responsive drug release at inflammatory sites (plaque microenvironments are typically weakly acidic). However, despite the extensive research on PDA as a drug carrier, its use for loading colchicine for specific treatment of atherosclerosis still faces significant challenges: First, it is necessary to solve how to efficiently and stably load high concentrations of colchicine into the PDA cavity while avoiding burst release; second, simple PDA nanoparticles lack active targeting of atherosclerotic plaques and are easily cleared by the body's immune system (such as the mononuclear phagocytic system); finally, a system capable of responding to the plaque microenvironment and intelligently releasing drugs is needed.

[0005] Therefore, developing a polydopamine nanomedicine delivery system that can efficiently load colchicine, possess active targeting and immune escape capabilities, and achieve controllable release at lesion sites is a pressing technical problem that needs to be solved in the field. Summary of the Invention

[0006] Based on the above analysis, the present invention aims to provide a method for preparing and applying colchicine-loaded polydopamine nanoparticles, in order to solve one of the problems of severe drug burst release, poor targeting, low bioavailability, and resulting toxic side effects in existing colchicine nanocarrier formulations.

[0007] The objective of this invention is mainly achieved through the following technical solutions:

[0008] This invention provides a method for preparing colchicine-loaded polydopamine nanoparticles, comprising the following steps:

[0009] S1: Prepare a macrophage cell membrane suspension for later use;

[0010] S2: Prepare platelet membrane suspension for later use;

[0011] S3: Mix macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles to obtain a first mixture, and then use an ultrasonic cell disruptor to sonicate the first mixture to obtain a membrane-coated first mixture.

[0012] S4: The first mixture treated with membrane coating is dispersed in colchicine aqueous solution to obtain the second mixture. The second mixture is ultrasonically treated using an ultrasonic cell disruptor, and colchicine is loaded into hollow polydopamine nanoparticles by electrostatic adsorption.

[0013] S5: At room temperature, centrifuge the second mixture after ultrasonic treatment, discard the supernatant and collect the precipitate, which is the colchicine-loaded polydopamine nanoparticles.

[0014] Further, step S1 includes:

[0015] Mouse macrophages J774A.1 were collected in 15 mL centrifuge tubes, centrifuged at 800-1200 rpm for 3-7 min at room temperature, and resuspended in PBS for counting.

[0016] Add 1 mL of membrane protein extraction reagent and 10 μL of benzosulfonyl fluoride to every 50 million mouse macrophages, resuspend the cells, incubate on ice for 8-12 min, and freeze and thaw twice in liquid nitrogen to ensure that 70-80% of the cells have no complete cell structure morphology when observed under a microscope.

[0017] Centrifuge the mixture at 4℃ and 4800-5200 rpm for 8-12 min to obtain the first supernatant and the first precipitate. Collect the first supernatant into a new centrifuge tube and discard the first precipitate.

[0018] Centrifuge the first supernatant at 13800-14200 rpm for 25-35 min at 4℃ to obtain the second supernatant and the second precipitate. The second precipitate is a macrophage membrane suspension. Resuspend and disperse it with PBS to obtain a macrophage membrane suspension, and store it at 4℃ for later use.

[0019] Further, step S2 includes:

[0020] Take 1 mL of whole blood from C57 mice and add it to a blood collection tube containing an anticoagulant;

[0021] At 4°C, whole blood from C57 mice was centrifuged at 1300-1700 rpm for 18-22 min to obtain the third supernatant and the third precipitate. The third supernatant was transferred to a new centrifuge tube, while the third precipitate was retained.

[0022] Centrifuge the third precipitate at 4800-5200 rpm for 18-22 min to obtain the fourth supernatant and the fourth precipitate. Discard the fourth supernatant and retain the fourth precipitate.

[0023] The fourth precipitate was washed three times with PBS pre-cooled to 4°C, and then the cells were resuspended in pre-cooled PBS to obtain a resuspended platelet suspension. The volume of pre-cooled PBS used for resuspending the cells was 1 / 5 of the total blood volume of the mouse.

[0024] The resuspended platelet suspension was repeatedly frozen and thawed three times at room temperature and -80°C. It was then centrifuged at 9800-10200 rpm for 8-12 minutes at 4°C. The supernatant was discarded after centrifugation, and the resulting precipitate was the platelet membrane. The precipitate was resuspended and dispersed with PBS to obtain the platelet membrane suspension, which was then stored at 4°C for later use.

[0025] Further, in step S3, the volume ratio of the macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles is (1-3):(1-3):1.

[0026] Furthermore, in step S3, the frequency of the ultrasonic treatment is 20-30kHz and the power is 90-110W.

[0027] Further, in step S4, the volume ratio of the first mixture of membrane coating treatment and colchicine aqueous solution is 1.5-2.

[0028] Furthermore, in step S4, the concentration of colchicine is 30-50 mg / mL.

[0029] Furthermore, in step S5, the centrifugation speed is 10000-12000 rpm and the centrifugation time is 8-12 min.

[0030] The present invention also provides a colchicine-loaded polydopamine nanoparticle prepared by the above preparation method.

[0031] The present invention also provides the application of the above-mentioned colchicine-loaded polydopamine nanoparticles in the preparation of drugs for treating atherosclerosis.

[0032] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0033] 1. This invention modifies hollow polydopamine nanoparticles with macrophage membrane suspension and platelet membrane suspension, and precisely controls the reaction conditions to encapsulate colchicine within the hollow polydopamine nanoparticles via electrostatic adsorption. This allows for the preparation of colchicine-loaded hollow polydopamine nanoparticles for the treatment of atherosclerosis. The colchicine-loaded hollow polydopamine nanoparticles prepared by this method exhibit a drug release rate controlled at 60%-70% within 48 hours. Compared to the rapid release of free drug (exceeding 80% within 24 hours), this method improves the loading efficiency of colchicine in hollow polydopamine nanoparticles and reduces non-specific adsorption and burst release phenomena, ensuring stable drug loading.

[0034] 2. This invention modifies hollow polydopamine nanoparticles with macrophage and platelet membrane suspensions and precisely controls the reaction conditions to encapsulate colchicine within the hollow polydopamine nanoparticles via electrostatic adsorption. The cell membrane biomimetic modification endows the nanoparticles with excellent active plaque targeting ability and immune escape function, significantly increasing the drug concentration at the lesion site, thereby enhancing efficacy and reducing toxic side effects on normal tissues. The nanomedicine can respond to the acidic microenvironment of the plaque, achieving intelligent and controllable drug release, further improving the precision of treatment.

[0035] 3. The colchicine-loaded hollow polydopamine nanomedicine prepared in this invention exhibits good biocompatibility and low toxicity. Targeting plaque sites results in higher local concentrations, effectively reducing the toxic side effects on healthy tissues. Furthermore, surface modification of the drug carrier helps reduce the immune system's recognition and clearance of the drug carrier, further improving the therapeutic window of colchicine and other drugs. HPDA itself possesses certain anti-inflammatory and antioxidant activities, which can synergistically enhance the anti-inflammatory effect of colchicine, thus improving the therapeutic efficacy against atherosclerosis.

[0036] 4. This invention validated the targeting and efficacy of colchicine-loaded polydopamine nanoparticles Col@PDA / PM in treating atherosclerosis in mice. Animal experiments showed that the nanomedicine did not cause significant abnormalities in routine blood tests, blood biochemical indicators, or pathological damage to major organs, demonstrating high safety and providing a basis for subsequent clinical trials.

[0037] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0038] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0039] Figure 1 The image shows the morphology of the hollow polydopamine nanoparticles prepared in Example 1 of this invention.

[0040] Figure 2 This is a schematic diagram of the method for preparing colchicine-loaded hollow polydopamine nanoparticles according to the present invention.

[0041] Figure 3 The image shows the morphology of the colchicine-loaded hollow polydopamine nanoparticles prepared in Example 1.

[0042] Figure 4 This is an in vivo imaging image of mice after injection of colchicine-loaded polydopamine nanoparticles in Example 2;

[0043] Figure 5 This is a statistical chart showing the white blood cell count, red blood cell count, lymphocyte count, and platelet count in the blood routine test indicators of each group of mice after the treatment in Example 2.

[0044] Figure 6 The image shows the organ indices of the liver, spleen, lungs, kidneys, and testes of mice in each group after treatment in Example 2.

[0045] Figure 7 HE staining images of liver, spleen, lung, kidney and testis tissues of mice in each group after treatment in Example 2;

[0046] Figure 8 This is a statistical chart of blood biochemical indicators of mice in each group after the treatment in Example 2;

[0047] Figure 9Oil Red staining images of mice in each group in Example 2 and their patch area statistics;

[0048] Figure 10 This is a statistical chart of four serum lipid parameters in mice after treatment in each group, as shown in Example 2.

[0049] Figure 11 This is an SDS-PAGE gel electrophoresis image of the colchicine-loaded hollow polydopamine nanoparticles prepared in Example 1.

[0050] Figure label:

[0051] 1-Control group; 2-Colchicine; 3-Hollow polydopamine; 4-Polydopamine nanoparticles loaded with colchicine; 5-Protein molecular weight standard; 6-Macrophage membrane; 7-Platelet membrane; ns-No statistical difference. Detailed Implementation

[0052] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0053] This invention provides a method for preparing hollow polydopamine nanoparticles loaded with colchicine, comprising the following steps:

[0054] S1: Preparation of macrophage membrane suspension

[0055] Mouse macrophages J774A.1 were collected in 15 mL centrifuge tubes, centrifuged at 800-1200 rpm for 3-7 min at room temperature, and resuspended in PBS for counting.

[0056] It should be noted that the mouse macrophage J774A.1 is a commonly used mouse macrophage cell line, derived from macrophages of C3H / He mice (an inbred mouse strain). After centrifugation, the mouse macrophages precipitate at the bottom of the centrifuge tube. After resuspending with PBS, a resuspended monolayer of mouse macrophage J774A.1 cells is obtained, which is used for subsequent cell counting.

[0057] Add 1 mL of membrane protein extraction reagent and 10 μL of 10 μL of methylsulfonyl fluoride to every 50 million mouse macrophages, resuspend the cells, incubate on ice for 8-12 min, and freeze and thaw twice in liquid nitrogen to ensure that 70-80% of the cells have no complete cell structure morphology when observed under a microscope.

[0058] It should be noted that after being placed in an ice bath and subjected to repeated freeze-thaw cycles in liquid nitrogen, the structure of mouse macrophages was destroyed, and components such as cell membranes and organelles were released into the solution, forming a mixture containing cell membrane proteins, organelle proteins, and other components.

[0059] Centrifuge the mixture at 4℃ and 4800-5200 rpm for 8-12 min to obtain the first supernatant and the first precipitate. Collect the first supernatant into a new centrifuge tube and discard the first precipitate.

[0060] It should be noted that the first supernatant mainly contains soluble components such as cell membrane proteins and organelle proteins, while the first precipitate mainly contains cell debris, cell nuclei, and incompletely broken cells. By centrifuging the mixture, larger particles such as cell debris, cell nuclei, and incompletely broken cells are precipitated, thereby removing these impurities and obtaining a relatively pure first supernatant.

[0061] Centrifuge the first supernatant at 13800-14200 rpm for 25-35 min at 4℃ to obtain the second supernatant and the second precipitate. The second precipitate is the macrophage membrane. Resuspend and disperse it with PBS to obtain the macrophage membrane suspension, and store it at 4℃ for later use.

[0062] It should be noted that cell membrane proteins have a large molecular weight and membrane structure. Therefore, after the first supernatant is centrifuged at high speed, cell membrane proteins will form a precipitate, while other soluble proteins and small molecules remain in the supernatant. The second precipitate is the macrophage membrane, which is rich in cell membrane proteins.

[0063] S2: Preparation of platelet membrane suspension

[0064] Take 1 mL of whole blood from C57 mice and add it to a blood collection tube containing an anticoagulant;

[0065] At 4°C, whole blood from C57 mice was centrifuged at 1300-1700 rpm for 18-22 min to obtain the third supernatant and the third precipitate. The third supernatant was transferred to a new centrifuge tube, while the third precipitate was retained.

[0066] Centrifuge the third precipitate at 4800-5200 rpm for 18-22 min to obtain the fourth supernatant and the fourth precipitate. Discard the fourth supernatant and retain the fourth precipitate.

[0067] It should be noted that after centrifuging the whole blood of C57 mice at 1300-1700 rpm for 18-22 minutes, the third supernatant mainly contains plasma (including serum proteins, coagulation factors, etc.), and the third precipitate mainly contains blood cells (including red blood cells, white blood cells, and platelets). This centrifugation step is mainly for the initial separation of blood cells and plasma. Then, the third precipitate is centrifuged at a higher speed to further separate platelets from other blood cells (white blood cells and red blood cells), while removing plasma components and other impurities that were not completely separated during the centrifugation of C57 mouse whole blood, making the platelets more concentrated and purified.

[0068] The fourth precipitate was washed three times with PBS pre-cooled to 4°C, and then the cells were resuspended in pre-cooled PBS to obtain a resuspended platelet suspension. The volume of pre-cooled PBS used for resuspending the cells was 1 / 5 of the total blood volume of the mouse.

[0069] It should be noted that the purpose of washing the fourth precipitate with PBS pre-cooled to 4°C is mainly to remove impurities and purify platelets through multiple washes; resuspending cells with pre-cooled PBS is mainly to adjust the platelet concentration.

[0070] The resuspended platelet suspension was repeatedly frozen and thawed three times at room temperature and -80°C. It was then centrifuged at 9800-10200 rpm for 8-12 minutes at 4°C. The supernatant was discarded after centrifugation, and the precipitate obtained was the platelet membrane. It was resuspended and dispersed with PBS to obtain the platelet membrane suspension, which was then stored at 4°C for later use.

[0071] It should be noted that repeated freeze-thaw cycles mainly disrupt the platelet cell membrane, releasing the cell contents; then, the platelet membrane is separated by centrifugation, resuspended and dispersed in PBS, and a resuspended platelet membrane suspension is obtained.

[0072] S3: Mix macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles to obtain a first mixture, and then use an ultrasonic cell disruptor to sonicate the first mixture to obtain a membrane-coated first mixture.

[0073] The ultrasonic treatment is performed at a frequency of 20-30 kHz and a power of 90-110 W. Through ultrasonic cavitation, the macrophage membrane and platelet membrane are fused and coated on the surface of hollow polydopamine nanoparticles to form cell membrane modified nanoparticles.

[0074] The volume ratio of macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles is (1-3):(1-3):1; preferably, the macrophage suspension is extracted and purified from 5000 cells and quantified in 1 mL; the platelet membrane suspension is extracted and purified from 1 mL of whole blood and quantified in 1 mL.

[0075] The concentration of hollow polydopamine nanoparticles is 1 mg / mL, and 1 mL is used.

[0076] S4: The first mixture of membrane coating treatment in step S3 is dispersed in colchicine aqueous solution to obtain the second mixture. The second mixture is ultrasonically treated using an ultrasonic cell disruptor, and colchicine is loaded into hollow polydopamine nanoparticles by electrostatic adsorption.

[0077] The ultrasonic treatment was performed at a frequency of 20-30 kHz and a power of 90-110 W. The second mixture was ultrasonically treated using an ultrasonic cell disruptor. During the ultrasonic treatment, colchicine was loaded into the hollow polydopamine nanoparticles and the membrane structure, ultimately achieving cell membrane encapsulation of hollow polydopamine nanoparticles and completing drug loading.

[0078] The volume ratio of the first mixture to the colchicine aqueous solution is 1.5-2, and the concentration of the colchicine aqueous solution is 30-50 mg / mL.

[0079] S5: At room temperature, centrifuge the second mixture after ultrasonic treatment, discard the supernatant and collect the precipitate, which is the colchicine-loaded polydopamine nanoparticles (named Col@PDA / PM).

[0080] The centrifugation speed is 10,000-12,000 rpm, and the centrifugation time is 8-12 min.

[0081] The preparation of colchicine-loaded polydopamine nanoparticles can be verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm whether the macrophage membrane and platelet membrane are successfully modified with hollow polydopamine nanoparticles.

[0082] The hollow polydopamine nanoparticles used in the above-mentioned method for preparing colchicine-loaded hollow polydopamine nanoparticles are prepared by the following method:

[0083] S1: Add 20×PBS solution, dimethyl sulfoxide (DMSO) and polydopamine hydrochloride to a beaker in sequence. React at room temperature. After the solution changes from colorless and transparent to brown, centrifuge at room temperature. The resulting precipitate is hollow polydopamine nanoparticles.

[0084] S2: The precipitate obtained in step S1 is washed three times with ultrapure water and ethanol to obtain pure hollow polydopamine nanoparticles.

[0085] Specifically, in step S1, the pH value of the 20×PBS solution is ≥8.5, the volume is 8-12 mL, the volume of dimethyl sulfoxide is 2-4 mL, the volume of polydopamine hydrochloride is 24-36 mg, the reaction time at room temperature is 10-15 h, after the solution changes from colorless and transparent to tea color, it is centrifuged at 10000-12000 rpm for 8-12 min at room temperature, and the resulting deposit is hollow polydopamine nanoparticles.

[0086] Hollow polydopamine nanoparticles with a particle size of approximately 180-220 nm were synthesized by using a mixture of alkaline PBS and DMSO as the reaction system and the precipitated phosphate crystals as the attachment core. This was achieved by oxidizing and polymerizing polydopamine hydrochloride in an alkaline environment with oxygen in the air to form black polydopamine.

[0087] It should be noted that hollow polydopamine nanoparticles possess strong hydrophilicity and surface modification capabilities, enabling them to interact with colchicine molecules through hydrogen bonds and electrostatic interactions, encapsulating colchicine within their structure. This prevents colchicine molecules from aggregating and precipitating in water, effectively improving the water solubility, stability, and bioavailability of colchicine. Furthermore, hollow polydopamine exhibits strong drug loading capacity and good biocompatibility, allowing colchicine to remain stable in vivo and effectively exert its pharmacological effects, overcoming the original problems of low water solubility and insufficient drug release associated with colchicine. Colchicine molecules can carry a positive charge at certain pH levels, while the phenolic hydroxyl / quinone groups enriched on the surface of hollow polydopamine can carry a negative charge under alkaline and neutral conditions. The two can bind together through strong electrostatic interactions. At the same time, the cavity structure of HPDA provides a physical encapsulation space for the drug. This dual mechanism of "electrostatic adsorption + physical encapsulation" achieves higher drug loading efficiency and more stable loading compared to liposomes or PLGA nanoparticles that rely solely on physical encapsulation, effectively preventing drug leakage and burst release during preparation and storage.

[0088] It should be noted that the necrotic core of atherosclerotic plaques contains a large number of macrophages. During plaque formation, vascular endothelial damage leads to the recruitment of platelets, creating a unique plaque microenvironment. To achieve targeted targeting of plaques, this invention employs a method of coating hollow polydopamine nanoparticles with a hybrid macrophage and platelet membrane. Macrophages, as important immune cells in the blood, benefit from coating with a macrophage membrane, which effectively enhances the camouflage effect of the hollow polydopamine nanoparticles, increasing immune escape and preventing clearance by immune cells in the circulating blood. Simultaneously, the platelet and macrophage membranes exhibit similar compatibility with macrophages within atherosclerotic plaques, increasing particle targeting and endocytosis efficiency. Therefore, this invention enhances the ability to target atherosclerotic plaques and improves the biocompatibility of hollow polydopamine nanoparticles in blood circulation by modifying hollow polydopamine nanoparticles with macrophage and platelet suspensions.

[0089] It should be noted that HPDA undergoes structural dissociation under acidic conditions, and the necrotic core and inflammatory region of atherosclerotic plaques constitute a typical weakly acidic microenvironment. The Col@PDA / PM prepared in this invention is stable in blood circulation (pH≈7.4) with slow drug release; however, when it accumulates in the acidic region of the plaque, the HPDA shell degrades rapidly, thereby achieving specific and responsive release of colchicine at the lesion site, improving treatment efficiency and reducing systemic exposure. The particle size of nanoparticles has a significant impact on drug release characteristics, especially in locally acidic inflammatory environments such as those associated with atherosclerosis. Generally, nanoparticles with excessively large sizes may have difficulty effectively penetrating the lesion site, while nanoparticles with excessively small sizes may lead to excessively rapid drug release, making it difficult to achieve a sustained and stable therapeutic effect. Therefore, the particle size typically needs to be controlled within the range of tens to hundreds of nanometers to ensure effective accumulation of nanoparticles at the inflammatory site while achieving slow and controllable drug release. The colchicine-loaded hollow polydopamine nanoparticles prepared by the method of this invention exhibit a drug release rate controlled at 60%-70% within 48 hours. Compared to the rapid release of free drug (exceeding 80% within 24 hours), this demonstrates that particle size regulation effectively improves the controllability of Col@PDA / PM drug release. Colchicine-loaded hollow polydopamine nanoparticles can respond to and promote targeted drug release under specific conditions of the acidic inflammatory environment in atherosclerosis. By adjusting the particle size of the Col@PDA / PM drug, continuous, stable, and controllable drug release can be achieved, thereby improving therapeutic efficacy and reducing the need for frequent dosing.

[0090] The targeting and efficacy of the prepared colchicine-loaded polydopamine nanoparticles Col@PDA / PM in the treatment of atherosclerosis were evaluated, including the following steps:

[0091] S1: Via ApoE - / - A mouse model of atherosclerosis was established by feeding mice a high-fat diet for 4-8 weeks.

[0092] S2: Inject Col@PDA / PM into atherosclerotic mice via intracanthal vein or tail vein, 2-3 times per week for 4 consecutive weeks;

[0093] S3: Col@PDA / PM was stained with DiR dye and its distribution in atherosclerotic mice was tracked by in vivo fluorescence imaging technology to evaluate the enrichment of Col@PDA / PM in atherosclerotic plaques and its binding to vascular cells.

[0094] S4: Evaluate the biosafety of Col@PDA / PM by combining routine blood tests, blood biochemistry, and pathological tissue sections of the heart, liver, kidney and testis of experimental animals;

[0095] S5: The effect of Col@PDA / PM on aortic plaques in mice was observed by staining the abdominal aorta of atherosclerotic mice with Oil Red dye.

[0096] S6: The effect of Col@PDA / PM on improving blood lipids in mice with atherosclerosis was evaluated by measuring blood lipid levels in mice.

[0097] It should be noted that in step S1, ApoE - / - (ApoE gene knockout) mice are a commonly used research model for atherosclerosis and cardiovascular disease; when ApoE... - / - Mice fed a high-fat diet (HFD) exhibit a range of pathological features similar to those of cardiovascular disease and metabolic syndrome in humans.

[0098] Specifically, in step S2, Col@PDA / PM is injected into atherosclerotic mice via the tail vein. The amount of Col@PDA / PM injected is related to the mouse's body weight, ranging from 8 to 12 μL / g of mouse body weight, and the concentration of the injected Col@PDA / PM is 0.2 to 0.4 mg / mL. Col@PDA / PM is dissolved in physiological saline for administration.

[0099] Specifically, in step S3, Col@PDA / PM is stained and labeled using DiR dye, and the distribution of Col@PDA / PM in atherosclerotic mice is tracked using in vivo fluorescence imaging technology to evaluate the enrichment degree of Col@PDA / PM in atherosclerotic plaques and its binding to vascular cells. This includes the following steps:

[0100] S31: Drug fluorescent labeling

[0101] Prepare a 10 μg / mL DiR working solution using PBS buffer; mix 1 mL of 1 mg / mL Col@PDA / PM suspension with 10 μL of DiR working solution; incubate in the dark for 2 h; centrifuge at 12000 rpm for 10 min at 4 °C, resuspend in PBS buffer, and wash three times; disperse Col@PDA / PM in physiological saline to a final concentration of 0.3 mg / mL.

[0102] S32: Animal administration

[0103] The labeled drug was administered to atherosclerotic mice via intracanthal vein injection. The animals were randomly divided into four groups of equal size, receiving one of the following: saline, colchicine solution (0.0067 mg / mL), PDA solution (0.3 mg / mL), or Col@PDA / PM solution (0.3 mg / mL), respectively. The dosage was 10 μL / g based on mouse body weight. Administration was twice weekly via intracanthal vein injection for four weeks.

[0104] S33: In vivo fluorescence imaging

[0105] Four hours after drug administration, mice were subjected to in vivo fluorescence imaging. Mice were placed in an anesthesia device and anesthetized with 3-4% isoflurane. The surface hair of the mice was removed, and the liver area of ​​the mice was blocked with black cardboard to prevent signal interference. The mice were placed in a small animal in vivo imaging device, and 1-2% isoflurane was maintained to keep the mice under continuous anesthesia. The distribution of each drug (i.e., physiological saline, colchicine, PDA, Col@PDA / PM) in the mice was observed.

[0106] S34: Analysis of Experimental Results

[0107] The intensity and distribution of fluorescence signals obtained by the in vivo fluorescence imaging system allow for a direct observation of the distribution of various drugs in mice.

[0108] Comparing the fluorescence signal intensity of atherosclerotic plaques with that of other normal tissues can help evaluate the enrichment level of various drugs in the plaque sites.

[0109] Specifically, step S4 includes: evaluating the biosafety of Col@PDA / PM by combining routine blood tests, blood biochemistry, and pathological tissue sections of the heart, liver, kidney, and testis of the experimental animals, specifically including:

[0110] S41: Complete Blood Count

[0111] After treatment, blood was collected from the eyeballs of mice according to the reference (Alix et al., (2018). Murine Pharmacokinetic Studies, Bio-protocol 8 (20): e3056.). The blood was injected into EDTA anticoagulant tubes and sent for testing immediately to detect the number of white blood cells, red blood cells, lymphocytes and platelets.

[0112] S42: Blood Biochemistry

[0113] The following references (The Clinical Chemistry of Laboratory Animals, 3rd Edition, Edited by David M. Kurtz and Gregory S. Travlos, First Published 18 October 2017, eBook, Published 18 October 2017, 1162 pages) were used to detect and analyze the blood biochemistry of mice after treatment. Blood was collected from the mice via the eyeballs. Whole blood samples were incubated at room temperature for 2 hours or overnight at 4°C, then centrifuged at 3000 rpm for 15 min at 2-8°C. The supernatant was immediately used for testing. Liver and kidney toxicity indicators were detected: alanine aminotransferase, aspartate aminotransferase, albumin, alkaline phosphatase, gamma-glutamyl transferase, total bile acids, urea, creatinine, and uric acid.

[0114] S43: Organ pathological sections and organ indices

[0115] After treatment, all major organs of the mice were removed, weighed, and visceral indices were calculated. After fixation, sections were prepared and stained with hematoxylin and eosin.

[0116] Hematoxylin-eosin (HE) staining: Hematoxylin is a basic dye that stains cell nuclei blue-black; eosin is an acidic dye that stains cytoplasm and other tissue structures pink. HE-stained tissue sections can be used to observe abnormal cell morphology and changes in tissue structure to determine whether the structure of tissues and organs is intact and to infer whether their function is impaired.

[0117] Specifically, step S5 includes: observing the effect of Col@PDA / PM on the abdominal aortic plaques of mice by staining the abdominal aorta of atherosclerotic mice with Oil Red dye, specifically including:

[0118] S51: Mix 6 parts of saturated Oil Red O staining solution with 4 parts of distilled water thoroughly and let stand overnight at 4°C. The next day, filter once with qualitative filter paper, and filter a second time after standing at 4°C for 24 hours to obtain Oil Red O working solution. Store at 4°C for later use.

[0119] After treatment, the mice were fixed on foam boards and perfused with physiological saline. The complete aorta was removed from the heart to the bifurcation of the iliac artery, and the surrounding adipose tissue was removed. The aorta was fixed with 4% paraformaldehyde for 24 hours, rinsed three times with PBS, and then longitudinally split. The aorta was immersed in 60% isopropanol for 5 minutes, transferred to Oil Red O working solution, and stained in the dark for 30 minutes. The mice were rinsed three times with 60% isopropanol and observed and photographed in PBS.

[0120] Specifically, step S6 includes: evaluating the effect of Col@PDA / PM on improving blood lipids in atherosclerotic mice by measuring blood lipid levels in mice, specifically including:

[0121] Blood was collected from the eyeballs of mice after treatment. Whole blood samples were left at room temperature for 2 hours or overnight at 4°C and then centrifuged at 3000 rpm for 15 minutes at 2-8°C. The supernatant was collected and tested immediately. Four lipid parameters were tested: triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein.

[0122] The method of the present invention will be described below with specific embodiments.

[0123] Example 1

[0124] This embodiment uses the hollow polydopamine nanoparticles of Example 1 to load colchicine, and prepares colchicine-loaded hollow polydopamine nanoparticles, including the following steps:

[0125] S1: Preparation of macrophage membrane suspension

[0126] Mouse macrophages J774A.1 were collected in 15 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min at room temperature, and resuspended in PBS for counting.

[0127] Add 1 mL of membrane protein extraction reagent and 10 μL of LPMSF to every 50 million mouse macrophages, incubate on ice for 10 min at room temperature, and repeat the freeze-thaw cycle twice in liquid nitrogen to ensure that 70-80% of the cells have no intact cell structure morphology when observed under a microscope. After the ice bath incubation and repeated freeze-thaw cycle in liquid nitrogen, the structure of mouse macrophages is destroyed, and components such as cell membranes and organelles are released into the solution, forming a mixture containing cell membrane proteins, organelle proteins and other components.

[0128] Centrifuge the mixture at 4℃ and 5000rpm for 10min to obtain the first supernatant and the first precipitate. Collect the first supernatant into a new centrifuge tube and discard the first precipitate.

[0129] Centrifuge the first supernatant at 14,000 rpm for 30 min at 4℃ to obtain the second supernatant and the second precipitate. The second precipitate is the macrophage membrane. Store at 4℃ for later use.

[0130] S2: Preparation of platelet membrane suspension

[0131] Take 1 mL of whole blood from C57 mice and add it to a blood collection tube containing an anticoagulant;

[0132] Whole blood from C57 mice was centrifuged at 1500 rpm for 20 min at 4℃ to obtain the third supernatant and the third precipitate. The third supernatant was transferred to a new centrifuge tube, while the third precipitate was retained.

[0133] Centrifuge the third precipitate at 5000 rpm for 20 min to obtain the fourth supernatant and the fourth precipitate. Discard the fourth supernatant and keep the fourth precipitate.

[0134] The fourth precipitate was washed three times with PBS pre-cooled to 4°C, and then the cells were resuspended in pre-cooled PBS to obtain a resuspended platelet suspension. The volume of pre-cooled PBS used for resuspending the cells was 1 / 5 of the mouse's whole blood volume (i.e., 0.2 mL).

[0135] The resuspended platelet suspension was repeatedly frozen and thawed three times at room temperature and -80°C. It was then centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded after centrifugation, and the resulting precipitate was the platelet membrane. The platelet membrane suspension was resuspended and dispersed with PBS and stored at 4°C for later use.

[0136] S3: Mix macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles to obtain a first mixture, and then use an ultrasonic cell disruptor to sonicate the first mixture to obtain a membrane-coated first mixture.

[0137] The ultrasonic treatment frequency was 25kHz and the power was 100W.

[0138] Macrophage suspension was prepared by purifying 5000 cells and quantifying in 1 mL; platelet membrane suspension was prepared by purifying 1 mL of whole blood and quantifying in 1 mL; hollow polydopamine nanoparticles had a concentration of 1 mg / mL and were used in 1 mL.

[0139] S4: The first mixture of membrane coating treatment in step S3 is dispersed in colchicine aqueous solution to obtain the second mixture. The second mixture is ultrasonically treated using an ultrasonic cell disruptor, and colchicine is loaded into hollow polydopamine nanoparticles by electrostatic adsorption.

[0140] The ultrasonic treatment frequency was 25kHz and the power was 100W.

[0141] The volume of the first mixture is 3 mL, the colchicine aqueous solution is 1 mL, and the concentration of the colchicine aqueous solution is 40 mg / mL.

[0142] S5: At room temperature, the ultrasonically treated mixture is centrifuged at 10,000 rpm for 10 min, the supernatant is discarded and the precipitate is collected. The precipitate is the colchicine-loaded polydopamine nanoparticles (named Col@PDA / PM).

[0143] The preparation of colchicine-loaded polydopamine nanoparticles was verified by SDS-PAGE to determine whether hollow polydopamine nanoparticles were successfully modified onto macrophage membranes and platelet membranes. Figure 11As shown in the SDS-PAGE gel electrophoresis image of Col@PDA / PM, the surface proteins of Col@PDA / PM are consistent with the protein expression on the surface of the original macrophage membrane and platelet membrane, indicating that the hollow polydopamine nanoparticles were successfully modified on the macrophage membrane and platelet membrane.

[0144] The hollow polydopamine used in this embodiment is prepared by the following method:

[0145] S1: Add 10 mL of 20×PBS solution with pH 8.5, 2 mL of dimethyl sulfoxide (DMSO), and 30 mg of polydopamine hydrochloride to a beaker in sequence. React at room temperature for 12 h. After the solution changes from colorless and transparent to brown, centrifuge at 12000 rpm for 10 min at room temperature. The resulting deposit is hollow polydopamine nanoparticles.

[0146] S2: The precipitate obtained in step S1 is washed three times with ultrapure water and ethanol to obtain pure hollow polydopamine nanoparticles.

[0147] Transmission electron microscopy (TEM) revealed that the obtained hollow polydopamine nanoparticles were regularly spherical with a particle size distribution of 180-200 nm and exhibited a distinct hollow structure. Figure 1 As shown.

[0148] Example 2

[0149] The colchicine-loaded polydopamine nanoparticles prepared in Example 1 were used in ApoE studies after 4 weeks of high-fat dieting. - / - Mice were used to evaluate the targeting and efficacy of Col@PDA / PM in the treatment of atherosclerosis.

[0150] Includes the following steps:

[0151] S1: Via ApoE - / - A mouse model of atherosclerosis was established by feeding mice a high-fat diet for 8 weeks.

[0152] Among them, ApoE was fed together - / - Twenty-eight mice were used to establish twenty-eight mouse models of atherosclerosis.

[0153] S2: Col@PDA / PM was injected into atherosclerotic mice via the inner canthal vein at a dose of 10 μL / g of mouse body weight, with a concentration of 0.3 mg / mL of Col@PDA / PM. Col@PDA / PM was dissolved in physiological saline for administration.

[0154] The mice were administered twice a week for four consecutive weeks. The mouse numbers, weights, and corresponding injection volumes are shown in Table 1.

[0155] Table 1 Information on mice injected with Col@PDA / PM

[0156]

[0157]

[0158] S3: Col@PDA / PM was stained with DiR dye and its distribution in atherosclerotic mice was tracked using in vivo fluorescence imaging to evaluate the enrichment of Col@PDA / PM in atherosclerotic plaques and its binding to vascular cells.

[0159] S31: Drug fluorescent labeling

[0160] Prepare a 10 μg / mL DiR working solution using PBS buffer; mix 1 mL of 1 mg / mL Col@PDA / PM suspension with 10 μL of DiR working solution; incubate in the dark for 2 h; centrifuge at 12000 rpm for 10 min at 4 °C, resuspend in PBS buffer, and wash three times; disperse Col@PDA / PM in physiological saline to a final concentration of 0.3 mg / mL.

[0161] S32: Animal administration

[0162] The labeled drug was administered to atherosclerotic mice via intracanthal vein injection. The animals were randomly divided into four groups of seven mice each, receiving physiological saline, colchicine solution (0.0067 mg / mL), PDA solution (0.3 mg / mL), or Col@PDA / PM solution (0.3 mg / mL), respectively. The dosage was 10 μL / g based on the mouse's body weight. Administration was twice weekly via intracanthal vein injection for a total of four weeks.

[0163] S33: In vivo fluorescence imaging

[0164] Four hours after drug administration, mice were subjected to in vivo fluorescence imaging. Mice were placed in an anesthesia device and anesthetized with 3-4% isoflurane. Hair was removed from the surface of the mice, and the liver area was blocked with black cardstock to prevent signal interference. Mice were then placed in a small animal in vivo imaging system, maintaining continuous anesthesia with 1-2% isoflurane. The distribution of each drug (one of physiological saline, colchicine, PDA, or Col@PDA / PM) in the mice was observed.

[0165] S34: Analysis of Experimental Results

[0166] The intensity and distribution of fluorescence signals obtained by the in vivo fluorescence imaging system allow for a direct observation of the distribution of various drugs in mice.

[0167] By comparing the fluorescence signal intensity of atherosclerotic plaques with that of other normal tissues, the enrichment degree of each drug at the plaque site can be evaluated.

[0168] Appendix Figure 4 The image shows an in vivo imaging of mice injected with Col@PDA / PM in this embodiment. As can be seen from the image, the drug molecules show a relatively obvious accumulation phenomenon in the atherosclerotic plaque area, indicating that the drug can specifically accumulate in the arterial plaque area, thereby increasing the local concentration of the drug.

[0169] Aside from plaque sites, the drug exhibits weak fluorescence intensity in normal blood vessels, organs, and other tissues, indicating that it has good targeting properties and can accumulate in plaque areas.

[0170] S4: Evaluate the biosafety of Col@PDA / PM by combining complete blood count, blood biochemistry, and pathological tissue sections of the heart, liver, kidney, and testis of experimental animals:

[0171] S41: Complete Blood Count

[0172] After treatment, blood was collected from the eyeballs of mice according to the reference (Alix et al., (2018). Murine Pharmacokinetic Studies, Bio-protocol 8 (20): e3056.). The blood was injected into EDTA anticoagulant tubes and sent for testing immediately to detect the number of white blood cells, red blood cells, lymphocytes and platelets.

[0173] S42: Blood Biochemistry

[0174] The following references (The Clinical Chemistry of Laboratory Animals, 3rd Edition, Edited by David M. Kurtz and Gregory S. Travlos, First Published 18 October 2017, eBook, Published 18 October 2017, 1162 pages) were used to detect and analyze the blood biochemistry of mice after treatment. Blood was collected from the mice via the eyeballs. Whole blood samples were incubated at room temperature for 2 hours or overnight at 4°C, then centrifuged at 3000 rpm for 15 min at 2-8°C. The supernatant was immediately used for testing. Liver and kidney toxicity indicators were detected: alanine aminotransferase, aspartate aminotransferase, albumin, alkaline phosphatase, gamma-glutamyl transferase, total bile acids, urea, creatinine, and uric acid.

[0175] S43: Organ pathological sections and organ indices

[0176] After treatment, all major organs of the mice were removed, weighed, and visceral indices were calculated. After fixation, sections were prepared and stained with hematoxylin and eosin.

[0177] Hematoxylin-eosin (HE) staining: Hematoxylin is a basic dye that stains cell nuclei blue-black; eosin is an acidic dye that stains cytoplasm and other tissue structures pink. HE-stained tissue sections can be used to observe abnormal cell morphology and changes in tissue structure to determine whether the structure of tissues and organs is intact and to infer whether their function is impaired.

[0178] Figure 5 This is a statistical chart showing the white blood cell count, red blood cell count, lymphocyte count, and platelet count in the blood routine test indicators of each group of mice after the treatment in this embodiment. Figure 6 This is an organ index graph of the liver, spleen, lungs, kidneys and testes of mice in each group after treatment in this embodiment; Figure 7 HE staining images of liver, spleen, lung, kidney and testis tissues of mice in each group after treatment in this embodiment; Figure 8 This is a statistical chart of blood biochemical indicators of mice in each group after the treatment in this embodiment;

[0179] from Figure 5 As can be seen, the mice's blood routine and blood biochemistry were within the normal range, and no abnormalities in the immune system were found, indicating that the drug did not cause systemic toxicity. Figure 8 The absence of abnormalities in blood biochemistry indicates that the drug did not cause liver or kidney damage, nor did it interfere with metabolic processes; Figure 6 and Figure 7 The pathological structures of the liver, spleen, lungs, kidneys, and testes remained intact, with no obvious damage, inflammation, or fibrosis observed. This demonstrates that the drug has no significant toxic effects on these vital organs, indicating that Col@PDA / PM did not cause significant toxic reactions in experimental animals and has good biosafety.

[0180] S5: The effect of Col@PDA / PM on abdominal aortic plaques in mice was observed by staining the abdominal aorta of atherosclerotic mice with Oil Red dye.

[0181] S51: Mix 6 parts of saturated Oil Red O staining solution with 4 parts of distilled water thoroughly and let stand overnight at 4°C. The next day, filter once with qualitative filter paper, and filter a second time after standing at 4°C for 24 hours to obtain Oil Red O working solution. Store at 4°C for later use.

[0182] After treatment, the mice were fixed on foam boards and perfused with physiological saline. The complete aorta was removed from the heart to the bifurcation of the iliac artery, and the surrounding adipose tissue was removed. The aorta was fixed with 4% paraformaldehyde for 24 hours, rinsed three times with PBS, and then longitudinally split. The aorta was immersed in 60% isopropanol for 5 minutes, transferred to Oil Red O working solution, and stained in the dark for 30 minutes. The mice were rinsed three times with 60% isopropanol and observed and photographed in PBS.

[0183] Appendix Figure 9 The images show Oil Red staining images of mice in each group in this embodiment and their plaque area statistics. The images show that the plaque area is smaller and lipid deposition is reduced, indicating that Col@PDA / PM has a good therapeutic effect on atherosclerosis.

[0184] S6: The effect of Col@PDA / PM on improving blood lipids in atherosclerotic mice was evaluated by measuring blood lipid levels in mice.

[0185] Blood was collected from the eyeballs of mice after treatment. Whole blood samples were left at room temperature for 2 hours or overnight at 4°C and then centrifuged at 3000 rpm for 15 minutes at 2-8°C. The supernatant was collected and tested immediately. Four lipid parameters were tested: triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein.

[0186] Appendix Figure 10 This is a statistical chart of four serum lipid parameters in mice after treatment in each group of this embodiment. Figure 10 As can be seen, Col@PDA / PM has a significant therapeutic effect on reducing blood lipids in atherosclerotic mice.

[0187] The evaluation of the targeting and efficacy of colchicine-loaded polydopamine nanoparticles (Col@PDA / PM) in treating atherosclerosis demonstrates that colchicine-loaded hollow polydopamine nanomedicine effectively inhibits local inflammation and plaque progression by precisely targeting atherosclerotic plaques. By inhibiting the activation of inflammatory cells and reducing cytokine secretion, the drug significantly reduces the pathological progression of atherosclerosis, slows plaque formation, promotes plaque stabilization, and prevents further aggravation of atherosclerosis.

[0188] Hollow polydopamine nanomedicine loaded with colchicine exhibits good biocompatibility and low toxicity, resulting in high local concentrations when targeted to plaque sites, thus effectively reducing the toxic side effects on healthy tissues. Furthermore, surface modification of the drug carrier helps reduce the recognition and clearance of the drug carrier by the immune system, further improving the therapeutic window of drugs such as colchicine.

[0189] The hollow polydopamine nanomedicine used in this invention has undergone rigorous in vivo safety evaluation, demonstrating good biodegradability and minimal accumulation in vital organs such as the liver and kidneys, significantly reducing drug toxicity and side effects. By detecting key biomarkers in vivo, its safety in the treatment of atherosclerosis can be confirmed, ensuring that the drug carrier has no significant negative impact on human health.

[0190] This method has been validated in laboratory mice. In subsequent clinical trials, the drug surface modification can be adjusted according to the specific condition of the patient to achieve personalized treatment. This highly tunable nanomedicine carrier system can provide a more flexible and precise treatment plan for atherosclerosis, adapting to the needs of different patients.

[0191] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing polydopamine nanoparticles loaded with colchicine, characterized in that, Includes the following steps: S1: Prepare a macrophage cell membrane suspension for later use; S2: Prepare platelet membrane suspension for later use; S3: Mix macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles to obtain a first mixture, and then use an ultrasonic cell disruptor to sonicate the first mixture to obtain a membrane-coated first mixture. S4: The first mixture treated with membrane coating is dispersed in colchicine aqueous solution to obtain the second mixture. The second mixture is ultrasonically treated using an ultrasonic cell disruptor, and colchicine is loaded into hollow polydopamine nanoparticles by electrostatic adsorption. S5: At room temperature, centrifuge the second mixture after ultrasonic treatment, discard the supernatant and collect the precipitate, which is the colchicine-loaded polydopamine nanoparticles.

2. The preparation method according to claim 1, characterized in that, Step S1 includes: Mouse macrophages J774A.1 were collected in 15 mL centrifuge tubes, centrifuged at 800-1200 rpm for 3-7 min at room temperature, and resuspended in PBS for counting. Add 1 mL of membrane protein extraction reagent and 10 μL of benzosulfonyl fluoride to every 50 million mouse macrophages, resuspend the cells, incubate on ice for 8-12 min, and freeze and thaw twice in liquid nitrogen to ensure that 70-80% of the cells have no complete cell structure morphology when observed under a microscope. Centrifuge the mixture at 4℃ and 4800-5200 rpm for 8-12 min to obtain the first supernatant and the first precipitate. Collect the first supernatant into a new centrifuge tube and discard the first precipitate. Centrifuge the first supernatant at 13800-14200 rpm for 25-35 min at 4℃ to obtain the second supernatant and the second precipitate. The second precipitate is a macrophage membrane suspension. Resuspend and disperse it with PBS to obtain a macrophage membrane suspension, and store it at 4℃ for later use.

3. The preparation method according to claim 2, characterized in that, Step S2 includes: Take 1 mL of whole blood from C57 mice and add it to a blood collection tube containing an anticoagulant; At 4°C, whole blood from C57 mice was centrifuged at 1300-1700 rpm for 18-22 min to obtain the third supernatant and the third precipitate. The third supernatant was transferred to a new centrifuge tube, while the third precipitate was retained. Centrifuge the third precipitate at 4800-5200 rpm for 18-22 min to obtain the fourth supernatant and the fourth precipitate. Discard the fourth supernatant and retain the fourth precipitate. The fourth precipitate was washed three times with PBS pre-cooled to 4°C, and then the cells were resuspended in pre-cooled PBS to obtain a resuspended platelet suspension. The volume of pre-cooled PBS used for resuspending the cells was 1 / 5 of the total blood volume of the mouse. The resuspended platelet suspension was repeatedly frozen and thawed three times at room temperature and -80°C. It was then centrifuged at 9800-10200 rpm for 8-12 minutes at 4°C. The supernatant was discarded after centrifugation, and the resulting precipitate was the platelet membrane. The precipitate was resuspended and dispersed with PBS to obtain the platelet membrane suspension, which was then stored at 4°C for later use.

4. The preparation method according to claim 3, characterized in that, In step S3, the volume ratio of the macrophage membrane suspension, platelet membrane suspension and hollow polydopamine nanoparticles is (1-3):(1-3):

1.

5. The preparation method according to claim 4, characterized in that, In step S3, the frequency of the ultrasonic treatment is 20-30kHz and the power is 90-110W.

6. The preparation method according to claim 5, characterized in that, In step S4, the volume ratio of the first mixture of membrane coating treatment and colchicine aqueous solution is 1.5-2.

7. The preparation method according to claim 6, characterized in that, In step S4, the concentration of colchicine is 30-50 mg / mL.

8. The preparation method according to claim 6, characterized in that, In step S5, the centrifugation speed is 10000-12000 rpm and the centrifugation time is 8-12 min.

9. A colchicine-loaded polydopamine nanoparticle prepared by any one of the preparation methods described in claims 1-8.

10. The use of the colchicine-loaded polydopamine nanoparticles of claim 9 in the preparation of a drug for treating atherosclerosis.

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